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Fabio Gentilini
Department of Veterinary Medical Sciences, Alma Mater Studiorum—University of Bologna, Ozzano dell’Emilia, 40064 Bologna, Italy

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Journal article
Published: 28 May 2021 in Viruses
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, China, in late 2019 and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) represents the gold standard for diagnostic assays even if it cannot precisely quantify viral RNA copies. Thus, we decided to compare qRT-PCR with digital polymerase chain reaction (dPCR), which is able to give an accurate number of RNA copies that can be found in a specimen. However, the aforementioned methods are not capable to discriminate if the detected RNA is infectious or not. For this purpose, it is necessary to perform an endpoint titration on cell cultures, which is largely used in the research field and provides a tissue culture infecting dose per mL (TCID50/mL) value. Both research and diagnostics call for a model that allows the comparison between the results obtained employing different analytical methods. The aim of this study is to define a comparison among two qRT-PCR protocols (one with preliminary RNA extraction and purification and an extraction-free qRT-PCR), a dPCR and a titration on cell cultures. The resulting correlations yield a faithful estimation of the total number of RNA copies and of the infectious viral burden from a Ct value obtained with diagnostic routine tests. All these estimations take into consideration methodological errors linked to the qRT-PCR, dPCR and titration assays.

ACS Style

Martina Brandolini; Francesca Taddei; Maria Marino; Laura Grumiro; Agata Scalcione; Maria Turba; Fabio Gentilini; Michela Fantini; Silvia Zannoli; Giorgio Dirani; Vittorio Sambri. Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management. Viruses 2021, 13, 1022 .

AMA Style

Martina Brandolini, Francesca Taddei, Maria Marino, Laura Grumiro, Agata Scalcione, Maria Turba, Fabio Gentilini, Michela Fantini, Silvia Zannoli, Giorgio Dirani, Vittorio Sambri. Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management. Viruses. 2021; 13 (6):1022.

Chicago/Turabian Style

Martina Brandolini; Francesca Taddei; Maria Marino; Laura Grumiro; Agata Scalcione; Maria Turba; Fabio Gentilini; Michela Fantini; Silvia Zannoli; Giorgio Dirani; Vittorio Sambri. 2021. "Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management." Viruses 13, no. 6: 1022.

Preprint content
Published: 29 April 2021
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Mutations in the receptor binding domain region of SARS-CoV-2 have been shown to impact the infectivity, pathogenicity and transmissibility of new variants. Even more worrisome, those mutations have the potential of causing immune escape, undermining the population immunity induced by ongoing mass vaccination programs. The massive parallel sequencing techniques have taken a lead role in the detection strategies of the new variants. Nevertheless, they are still cumbersome and labour-demanding. As a matter of fact, there is an urgent need for novel strategies and techniques aimed at the surveillance of the active emergence and spread of the variants of concern. Two PCRs were designed to target the binding domain region, spanning residues N417 through N501 of the Spike protein. Furthermore, very rapid, affordable and straightforward Denaturing High-Performance Liquid Chromatography screening was set up. The screening consisted of mixing the unknown sample with a standard sample of a known sequence, denaturing at high temperature for two minutes, renaturing for 15 minutes followed by a 2-minute run using the WAVE DHPLC system to detect the heteroduplexes which invariably originate whenever the unknown sample has a nucleotide difference with respect to the standard used. The workflow was able to readily detect new variants including the P.1 and the B.1.585 strains at a very affordable cost. This approach has the potential of greatly expediting surveillance of the SARS-CoV-2 variants.

ACS Style

Maria E Turba; Domenico Mion; Stavros Papadimitriou; Francesca Taddei; Giorgio Dirani; Vittorio Sambri; Fabio Gentilini. Affordable and time-effective high throughput screening of SARS-CoV-2 variants using Denaturing High-Performance Liquid Chromatography analysis. 2021, 1 .

AMA Style

Maria E Turba, Domenico Mion, Stavros Papadimitriou, Francesca Taddei, Giorgio Dirani, Vittorio Sambri, Fabio Gentilini. Affordable and time-effective high throughput screening of SARS-CoV-2 variants using Denaturing High-Performance Liquid Chromatography analysis. . 2021; ():1.

Chicago/Turabian Style

Maria E Turba; Domenico Mion; Stavros Papadimitriou; Francesca Taddei; Giorgio Dirani; Vittorio Sambri; Fabio Gentilini. 2021. "Affordable and time-effective high throughput screening of SARS-CoV-2 variants using Denaturing High-Performance Liquid Chromatography analysis." , no. : 1.

Case report
Published: 19 February 2020 in BMC Veterinary Research
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Background Gain-of-function mutations in KIT are driver events of oncogenesis in mast cell tumours (MCTs) affecting companion animals. Somatic mutations of KIT determine the constitutive activation of the tyrosine kinase receptor leading to a worse prognosis and a shorter survival time than MCTs harbouring wild-type KIT. However, canine MCTs carrying KIT somatic mutations generally respond well to tyrosine kinase inhibitors; hence their presence represents a predictor of treatment effectiveness, and its detection allows implementing a stratified medical approach. Despite this, veterinary oncologists experience treatment failures, even with targeted therapies whose cause cannot be elucidated. The first case of an MCT-affected dog caused by a secondary mutation in the tyrosine kinase domain responsible for resistance has recently been reported. The knowledge of this and all the other mutations responsible for resistance would allow the effective bedside implementation of a deeply stratified and more effective medical approach. Case presentation The second case of a canine MCT carrying a different resistance mutation is herein described. The case was characterised by aggressive behaviour and early metastasis unresponsive to both vinblastine- and masitinib-based treatments. Molecular profiling of the tumoural masses revealed two different mutations; other than the already known activating mutation p.Asn508Ile in KIT exon 9, which is tyrosine kinase inhibitor-sensitive, a nearly adjacent secondary missense mutation, p.Ala510Val, which had never before been described, was detected. In vitro transfection experiments showed that the secondary mutation did not cause the constitutive activation by itself but played a role in conferring resistance to masitinib. Conclusions This study highlighted the importance of the accurate molecular profiling of an MCT in order to improve understanding of the molecular mechanism underlying tumourigenesis and reveal chemoresistance in MCTs for more effective therapies. The detection of the somatic mutations responsible for resistance should be included in the molecular screening of MCTs, and a systematic analysis of all the cases characterised by unexpected refractoriness to therapies should be investigated in depth at both the genetic and the phenotypic level.

ACS Style

Fabio Gentilini; Maria Elena Turba; Claire Dally; Masamine Takanosu; Sena Kurita; Makoto Bonkobara. The secondary KIT mutation p.Ala510Val in a cutaneous mast cell tumour carrying the activating mutation p.Asn508Ile confers resistance to masitinib in dogs. BMC Veterinary Research 2020, 16, 1 -9.

AMA Style

Fabio Gentilini, Maria Elena Turba, Claire Dally, Masamine Takanosu, Sena Kurita, Makoto Bonkobara. The secondary KIT mutation p.Ala510Val in a cutaneous mast cell tumour carrying the activating mutation p.Asn508Ile confers resistance to masitinib in dogs. BMC Veterinary Research. 2020; 16 (1):1-9.

Chicago/Turabian Style

Fabio Gentilini; Maria Elena Turba; Claire Dally; Masamine Takanosu; Sena Kurita; Makoto Bonkobara. 2020. "The secondary KIT mutation p.Ala510Val in a cutaneous mast cell tumour carrying the activating mutation p.Asn508Ile confers resistance to masitinib in dogs." BMC Veterinary Research 16, no. 1: 1-9.

Original article
Published: 16 January 2020 in Veterinary and Comparative Oncology
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Platelet‐derived growth factor signaling pathways play a fundamental role in inducing and sustaining the proliferative and prosurvival stimuli in canine osteosarcomas (cOSAs). The increased expression of platelet‐derived growth factor receptors (PDGFRs) α and β, and their cognate ligands, were almost invariably observed in cOSAs and OSA‐derived cell lines. In particular, overexpression of PDGFRβ‐mediated signaling pathways was found in both the tumour microenvironment, where it drives stromal cell recruitment, and in neoangiogenesis, such as in tumour cells where it triggers aberrant proliferation, migration and local invasion. The majority of the pathological consequences of PDGFRβ signaling are due to aberrant expression. In fact, epigenetic dysregulation of oncogenes throughout demethylation of their promoter has emerged as a pivotal mechanism driving oncogenesis. The aim of this study was to assess the methylation status of the PDGFRβ promoter and to clarify its role in modulating the expression of the tyrosine kinase receptor in canine osteosarcoma. The CpG island of the PDGFRβ promoter was identified using a mixed in silico and experimental approach, and a method based upon the Methylation‐Sensitive High‐Resolution Melting assay for quantitatively and precisely assessing the methylation status of the promoter was then set up. The method herein described was then exploited to assess the methylation status of the promoter in a case series of cOSAa. COSAs consistently but variably expressed PDGFRβ. However, the promoter was almost completely demethylated, and its methylation status did not correlate with the expression levels. This finding supported the hypothesis that post‐transcriptional regulatory mechanisms may act in cOSAs. This article is protected by copyright. All rights reserved.

ACS Style

Fabio Gentilini; Ombretta Capitani; Debora Tinto; Antonella Rigillo; Silvia Sabattini; Giuliano Bettini; Elena Turba Maria. Assessment of PDGFRβ promoter methylation in canine osteosarcoma using methylation‐sensitive high‐resolution melting analysis. Veterinary and Comparative Oncology 2020, 18, 484 -493.

AMA Style

Fabio Gentilini, Ombretta Capitani, Debora Tinto, Antonella Rigillo, Silvia Sabattini, Giuliano Bettini, Elena Turba Maria. Assessment of PDGFRβ promoter methylation in canine osteosarcoma using methylation‐sensitive high‐resolution melting analysis. Veterinary and Comparative Oncology. 2020; 18 (4):484-493.

Chicago/Turabian Style

Fabio Gentilini; Ombretta Capitani; Debora Tinto; Antonella Rigillo; Silvia Sabattini; Giuliano Bettini; Elena Turba Maria. 2020. "Assessment of PDGFRβ promoter methylation in canine osteosarcoma using methylation‐sensitive high‐resolution melting analysis." Veterinary and Comparative Oncology 18, no. 4: 484-493.

Multicenter study
Published: 12 September 2019 in The Veterinary Journal
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Epilepsy is the most common chronic neurological disorder in dogs. Approximately 20-30% of dogs do not achieve satisfactory seizure control with two or more anti-epileptic drugs at appropriate dosages. This condition, defined as refractory epilepsy, is a multifactorial condition involving both acquired and genetic factors. The P glycoprotein might play and important role in the pathophysiological mechanism and it is encoded by the ABCB1 gene. An association between a single nucleotide variation of the ABCB1 gene (c.-6-180T>G) and phenobarbital resistance has previously been reported in a Border collie population with idiopathic epilepsy. To date, the presence and relevance of this polymorphism has not been assessed in other breeds. A multicentre retrospective, case-control study was conducted to investigate associations between ABCB1 c.-6-180T>G, clinical variables, and refractoriness in a multi-breed population of dogs with refractory idiopathic epilepsy. A secondary aim was to evaluate the possible involvement of the ABCB1 c.-6-180T>G single nucleotide variation this population. Fifty-two refractory and 50 responsive dogs with idiopathic epilepsy were enrolled. Of these, 45 refractory and 50 responsive (control) dogs were genotyped. The G allele was found in several breeds, but there was no evidence of association with refractoriness (P=0.69). The uncertain role of the c.-6-180T>G variation was further suggested by an association between the T/T genotype with both refractoriness and responsiveness in different breeds. Furthermore, high seizure density (cluster seizure) was the main clinical risk factor for refractory idiopathic epilepsy (P=0.003).

ACS Style

T. Gagliardo; G. Gandini; A. Gallucci; M. Menchetti; E. Bianchi; M.E. Turba; A. Cauduro; D.S. Corlazzoli; S. Gianni; M. Baroni; M. Bernardini; F. Gentilini. ABCB1 c.-6-180T>G polymorphism and clinical risk factors in a multi-breed cohort of dogs with refractory idiopathic epilepsy. The Veterinary Journal 2019, 253, 105378 .

AMA Style

T. Gagliardo, G. Gandini, A. Gallucci, M. Menchetti, E. Bianchi, M.E. Turba, A. Cauduro, D.S. Corlazzoli, S. Gianni, M. Baroni, M. Bernardini, F. Gentilini. ABCB1 c.-6-180T>G polymorphism and clinical risk factors in a multi-breed cohort of dogs with refractory idiopathic epilepsy. The Veterinary Journal. 2019; 253 ():105378.

Chicago/Turabian Style

T. Gagliardo; G. Gandini; A. Gallucci; M. Menchetti; E. Bianchi; M.E. Turba; A. Cauduro; D.S. Corlazzoli; S. Gianni; M. Baroni; M. Bernardini; F. Gentilini. 2019. "ABCB1 c.-6-180T>G polymorphism and clinical risk factors in a multi-breed cohort of dogs with refractory idiopathic epilepsy." The Veterinary Journal 253, no. : 105378.

Research article
Published: 04 September 2019 in PLOS ONE
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Inherited bleeding disorders including abnormalities of platelet number and function rarely occur in a variety of dog breeds, but are probably underdiagnosed. Genetically characterized canine forms of platelet disorders provide valuable large animal models for understanding similar platelet disorders in people. Breed-specific disease associated genetic variants in only eight different genes are known to cause intrinsic platelet disorders in dogs. However, the causative genetic variant in many dog breeds has until now remained unknown. Four cases of a mild to severe bleeding disorder in Cocker Spaniel dogs are herein presented. The affected dogs showed a platelet adhesion defect characterized by macrothrombocytopenia with variable platelet counts resembling human Bernard-Soulier syndrome (BSS). Furthermore, the lack of functional GPIb-IX-V was demonstrated by immunocytochemistry. Whole genome sequencing of one affected dog and visual inspection of the candidate genes identified a deletion in the glycoprotein IX platelet (GP9) gene. The GP9 gene encodes a subunit of a platelet surface membrane glycoprotein complex; this functions as a receptor for von Willebrand factor, which initiates the maintenance of hemostasis after injury. Variants in human GP9 are associated with Bernard-Soulier syndrome, type C. The deletion spanned 2460 bp, and included a significant part of the single coding exon of the canine GP9 gene on dog chromosome 20. The variant results in a frameshift and premature stop codon which is predicted to truncate almost two-thirds of the encoded protein. PCR-based genotyping confirmed recessive inheritance. The homozygous variant genotype seen in affected dogs did not occur in 98 control Cocker Spaniels. Thus, it was concluded that the structural variant identified in the GP9 gene was most likely causative for the BSS-phenotype in the dogs examined. These findings provide the first large animal GP9 model for this group of inherited platelet disorders and greatly facilitate the diagnosis and identification of affected and/or normal carriers in Cocker Spaniels.

ACS Style

Fabio Gentilini; Maria Elena Turba; Fiorella Giancola; Roberto Chiocchetti; Chiara Bernardini; Markéta Dajbychova; Vidhya Jagannathan; Michaela Drögemüller; Cord Drögemüller. A large deletion in the GP9 gene in Cocker Spaniel dogs with Bernard-Soulier syndrome. PLOS ONE 2019, 14, e0220625 .

AMA Style

Fabio Gentilini, Maria Elena Turba, Fiorella Giancola, Roberto Chiocchetti, Chiara Bernardini, Markéta Dajbychova, Vidhya Jagannathan, Michaela Drögemüller, Cord Drögemüller. A large deletion in the GP9 gene in Cocker Spaniel dogs with Bernard-Soulier syndrome. PLOS ONE. 2019; 14 (9):e0220625.

Chicago/Turabian Style

Fabio Gentilini; Maria Elena Turba; Fiorella Giancola; Roberto Chiocchetti; Chiara Bernardini; Markéta Dajbychova; Vidhya Jagannathan; Michaela Drögemüller; Cord Drögemüller. 2019. "A large deletion in the GP9 gene in Cocker Spaniel dogs with Bernard-Soulier syndrome." PLOS ONE 14, no. 9: e0220625.

Journal article
Published: 01 November 2017 in Journal of Veterinary Behavior
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ACS Style

Marika Menchetti; Gualtiero Gandini; Antonella Gallucci; Giorgia Della Rocca; Lara Matiasek; Kaspar Matiasek; Fabio Gentilini; Marco Rosati. Approaching phantom complex after limb amputation in the canine species. Journal of Veterinary Behavior 2017, 22, 24 -28.

AMA Style

Marika Menchetti, Gualtiero Gandini, Antonella Gallucci, Giorgia Della Rocca, Lara Matiasek, Kaspar Matiasek, Fabio Gentilini, Marco Rosati. Approaching phantom complex after limb amputation in the canine species. Journal of Veterinary Behavior. 2017; 22 ():24-28.

Chicago/Turabian Style

Marika Menchetti; Gualtiero Gandini; Antonella Gallucci; Giorgia Della Rocca; Lara Matiasek; Kaspar Matiasek; Fabio Gentilini; Marco Rosati. 2017. "Approaching phantom complex after limb amputation in the canine species." Journal of Veterinary Behavior 22, no. : 24-28.

Journal article
Published: 04 December 2016 in Animal Genetics
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Degenerative myelopathy is a severe and progressive neurodegenerative disease and, in the majority of breeds, is associated with the c.118G>A substitution in exon 2 of the canine superoxide dismutase 1 (SOD1) gene. Our laboratories have been engaged in determining the cause of many discordant findings between the parental and the offspring genotypes found by different laboratories. In both cases, the discordant findings refer to actual heterozygous dogs that had been typed as homozygous for the variant allele. To that aim, the genomic context of the causative variant was investigated in two Hovawart dogs. An insertion of 54 nucleotides composed of a poly-T stretch and 15 nucleotides containing the duplication of the exon 2–intron 2 junction was found. The insertion was responsible for the partial mismatch of the reverse primer used for a direct sequencing assay. The mismatch hampered the amplification of the corresponding allele and caused an evident drop-out effect. The insertion is in complete linkage disequilibrium with the c.118G allele. The allele containing the insertion was highly prevalent in Hovawart dogs, accounting for the 26.6% of allele frequency. The insertion was also found in other unrelated breeds such as Rough Collies and Standard Poodles. In conclusion, the study illustrates the importance of correctly designing the primers to avoid inaccurate genotyping of the degenerative myelopathy causative variant in exon 2 of the SOD1 gene.

ACS Style

M. E. Turba; R. Loechel; E. Rombolà; Gualtiero Gandini; Fabio Gentilini. Evidence of a genomic insertion in intron 2 of SOD1 causing allelic drop-out during routine diagnostic testing for canine degenerative myelopathy. Animal Genetics 2016, 48, 365 -368.

AMA Style

M. E. Turba, R. Loechel, E. Rombolà, Gualtiero Gandini, Fabio Gentilini. Evidence of a genomic insertion in intron 2 of SOD1 causing allelic drop-out during routine diagnostic testing for canine degenerative myelopathy. Animal Genetics. 2016; 48 (3):365-368.

Chicago/Turabian Style

M. E. Turba; R. Loechel; E. Rombolà; Gualtiero Gandini; Fabio Gentilini. 2016. "Evidence of a genomic insertion in intron 2 of SOD1 causing allelic drop-out during routine diagnostic testing for canine degenerative myelopathy." Animal Genetics 48, no. 3: 365-368.

Journal article
Published: 14 June 2016 in Animal Genetics
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ACS Style

Fabio Gentilini; Elisa Zambon; Danilo Mancini; Maria Elena Turba. A nonsense mutation in themyeloperoxidasegene is responsible for hereditary myeloperoxidase deficiency in an Italian hound dog. Animal Genetics 2016, 47, 632 -633.

AMA Style

Fabio Gentilini, Elisa Zambon, Danilo Mancini, Maria Elena Turba. A nonsense mutation in themyeloperoxidasegene is responsible for hereditary myeloperoxidase deficiency in an Italian hound dog. Animal Genetics. 2016; 47 (5):632-633.

Chicago/Turabian Style

Fabio Gentilini; Elisa Zambon; Danilo Mancini; Maria Elena Turba. 2016. "A nonsense mutation in themyeloperoxidasegene is responsible for hereditary myeloperoxidase deficiency in an Italian hound dog." Animal Genetics 47, no. 5: 632-633.

Brief communication
Published: 01 December 2015 in Acta Veterinaria Scandinavica
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Platelet-rich plasma (PRP) commonly refers to blood products which contain a higher platelet (PLT) concentration as compared to normal plasma. Autologous PRP has been shown to be safe and effective in promoting the natural processes of soft tissue healing or reconstruction in humans and horses. Variability in PLT concentration has been observed in practice between PRP preparations from different patients or from the same individual under different conditions. A change in PLT concentration could modify PRP efficacy in routine applications. The aim of this study was to test the influence of environmental, individual and agonistic variables on the PLT concentration of PRP in horses. Six healthy Standardbred mares were exposed to six different variables with a one-week washout period between variables, and PRP was subsequently obtained from each horse. The variables were time of withdrawal during the day (morning/evening), hydration status (overhydration/dehydration) treatment with anti-inflammatory drugs and training periods on a treadmill. The platelet concentration was significantly higher in horses treated with a non-steroidal anti-inflammatory drug (P = 0.03). The leukocyte concentration increased 2-9 fold with respect to whole blood in the PRP which was obtained after exposure to all the variable considered. Environmental variation in platelet concentration should be taken into consideration during PRP preparation.

ACS Style

Riccardo Rinnovati; Noemi Romagnoli; Fabio Gentilini; Carlotta Lambertini; Alessandro Spadari. The influence of environmental variables on platelet concentration in horse platelet-rich plasma. Acta Veterinaria Scandinavica 2015, 58, 45 .

AMA Style

Riccardo Rinnovati, Noemi Romagnoli, Fabio Gentilini, Carlotta Lambertini, Alessandro Spadari. The influence of environmental variables on platelet concentration in horse platelet-rich plasma. Acta Veterinaria Scandinavica. 2015; 58 (1):45.

Chicago/Turabian Style

Riccardo Rinnovati; Noemi Romagnoli; Fabio Gentilini; Carlotta Lambertini; Alessandro Spadari. 2015. "The influence of environmental variables on platelet concentration in horse platelet-rich plasma." Acta Veterinaria Scandinavica 58, no. 1: 45.

Comparative study
Published: 08 October 2015 in Journal of Veterinary Diagnostic Investigation
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Leptospires are excreted in the urine of infected animals, and the prompt detection of leptospiral DNA using polymerase chain reaction (PCR) is increasingly being used. However, contradictory data has emerged concerning the diagnostic accuracy of the most popular PCR assays that target either the 16S ribosomal RNA ( rrs) or the subsurface lipoprotein ( LipL32) genes. In order to clarify the effect of the gene target, a novel hydrolysis probe–based, quantitative real-time PCR (qPCR) assay targeting the LipL32 gene was developed, validated, and then compared directly to the previously described rrs hydrolysis probe–based qPCR using a convenience collection of canine urine samples. The novel LipL32 qPCR assay was linear from 5.9 × 106 to 59 genome equivalents per reaction. Both the LipL32 and the rrs qPCR assays showed a limit of detection of 10 target copies per reaction indicating an approximately equivalent analytical sensitivity. Both assays amplified all 20 pathogenic leptospiral strains tested but did not amplify a representative collection of bacteria commonly found in voided canine urine. When the field samples were assayed, 1 and 5 out of 184 samples yielded an amplification signal in the LipL32 and rrs assays, respectively. Nevertheless, when the limit of detection was considered as the cutoff for interpreting findings, the 4 discordant cases were judged as negative. In conclusion, our study confirmed that both LipL32 and rrs are suitable targets for qPCR for the detection of leptospiral DNA in canine urine. However, the rrs target requires the mandatory use of a cutoff value in order to correctly interpret spurious amplifications.

ACS Style

Fabio Gentilini; Renato Giulio Zanoni; Elisa Zambon; Maria Elena Turba. A comparison of two real-time polymerase chain reaction assays using hybridization probes targeting either 16S ribosomal RNA or a subsurface lipoprotein gene for detecting leptospires in canine urine. Journal of Veterinary Diagnostic Investigation 2015, 27, 696 -703.

AMA Style

Fabio Gentilini, Renato Giulio Zanoni, Elisa Zambon, Maria Elena Turba. A comparison of two real-time polymerase chain reaction assays using hybridization probes targeting either 16S ribosomal RNA or a subsurface lipoprotein gene for detecting leptospires in canine urine. Journal of Veterinary Diagnostic Investigation. 2015; 27 (6):696-703.

Chicago/Turabian Style

Fabio Gentilini; Renato Giulio Zanoni; Elisa Zambon; Maria Elena Turba. 2015. "A comparison of two real-time polymerase chain reaction assays using hybridization probes targeting either 16S ribosomal RNA or a subsurface lipoprotein gene for detecting leptospires in canine urine." Journal of Veterinary Diagnostic Investigation 27, no. 6: 696-703.

Journal article
Published: 01 July 2015 in The Veterinary Journal
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Equine colic may be associated with an acute phase response (APR). Measurement of acute phase proteins (APPs) allows the detection of an APR and may help clinicians in monitoring the disease; however, the role of APPs in colic is unclear. This study aimed to evaluate the clinical usefulness of serum amyloid A (SAA), haptoglobin and ferritin in combination with an extended clinicopathological profile in equine colic. The medical records of 54 horses were retrospectively selected. Horses were grouped based on outcome (survivors vs. non-survivors), diagnosis (ischaemic/strangulating vs. non-ischaemic/non-strangulating), and treatment (medical treatment vs. surgery). Laboratory data were compared, and a logistic regression analysis was performed for outcome prediction upon admission. A high percentage of horses had abnormal SAA (29/54), haptoglobin (20/54), and ferritin (31/54) concentrations. In particular, haptoglobin was below the reference interval in 13/54 horses. Non-survivors had significantly decreased haptoglobin and increased ferritin concentrations compared with survivors. The ischaemic/strangulating group had significantly increased creatinine and ferritin and decreased haptoglobin concentrations compared with the non-ischaemic/non-strangulating group. Creatinine was the only significant predictor of mortality in the regression analysis. In conclusion, APPs including SAA, haptoglobin, and ferritin combined with clinicopathological variables may help clinicians to understand the pathogenesis of APR and underline potential complications of equine colic. The reduction in haptoglobin concentration may suggest haemolysis or muscle fibre damage; ferritin may indicate alteration in iron metabolism and tissue damage. Further prospective studies are needed to assess diagnostic and prognostic values of APPs in colic horses.

ACS Style

Francesco Dondi; Robert M. Lukacs; Fabio Gentilini; Riccardo Rinnovati; Alessandro Spadari; Noemi Romagnoli. Serum amyloid A, haptoglobin, and ferritin in horses with colic: Association with common clinicopathological variables and short-term outcome. The Veterinary Journal 2015, 205, 50 -55.

AMA Style

Francesco Dondi, Robert M. Lukacs, Fabio Gentilini, Riccardo Rinnovati, Alessandro Spadari, Noemi Romagnoli. Serum amyloid A, haptoglobin, and ferritin in horses with colic: Association with common clinicopathological variables and short-term outcome. The Veterinary Journal. 2015; 205 (1):50-55.

Chicago/Turabian Style

Francesco Dondi; Robert M. Lukacs; Fabio Gentilini; Riccardo Rinnovati; Alessandro Spadari; Noemi Romagnoli. 2015. "Serum amyloid A, haptoglobin, and ferritin in horses with colic: Association with common clinicopathological variables and short-term outcome." The Veterinary Journal 205, no. 1: 50-55.

Journal article
Published: 01 October 2014 in Research in Veterinary Science
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On growth performances, blood parameters, systemic cytokine levels, cell stress markers and reactivity\ud of immune system of weaned pigs.\ud Growth performance was not affected by OTA consumption even if OTA levels increased in plasma,\ud kidney and liver. OTA diminished the protein content in the serum and increased levels of TNF-alpha\ud and IL-10 in plasma. HO-1 mRNA, indicative for cells stress, was decreased in the kidney but increased\ud in the liver. Additionally, whole blood of the animals of the OTA-group showed a decreased capacity to\ud respond with cytokine expression (mRNA and protein) to ex vivo challenge with LPS. In conclusion our\ud findings indicate that chronic ingestion with OTA-contaminated feed, even at low level, is hazardous for\ud the animal and virtually for human health, pig being an excellent model for human

ACS Style

Chiara Bernardini; Ester Grilli; Johanna Catharina Duvigneau; Augusta Zannoni; Benedetta Tugnoli; Fabio Gentilini; Terenzio Bertuzzi; Silvia Spinozzi; Cecilia Camborata; Maria Laura Bacci; Andrea Piva; Monica Forni. Cellular stress marker alteration and inflammatory response in pigs fed with an ochratoxin contaminated diet. Research in Veterinary Science 2014, 97, 244 -250.

AMA Style

Chiara Bernardini, Ester Grilli, Johanna Catharina Duvigneau, Augusta Zannoni, Benedetta Tugnoli, Fabio Gentilini, Terenzio Bertuzzi, Silvia Spinozzi, Cecilia Camborata, Maria Laura Bacci, Andrea Piva, Monica Forni. Cellular stress marker alteration and inflammatory response in pigs fed with an ochratoxin contaminated diet. Research in Veterinary Science. 2014; 97 (2):244-250.

Chicago/Turabian Style

Chiara Bernardini; Ester Grilli; Johanna Catharina Duvigneau; Augusta Zannoni; Benedetta Tugnoli; Fabio Gentilini; Terenzio Bertuzzi; Silvia Spinozzi; Cecilia Camborata; Maria Laura Bacci; Andrea Piva; Monica Forni. 2014. "Cellular stress marker alteration and inflammatory response in pigs fed with an ochratoxin contaminated diet." Research in Veterinary Science 97, no. 2: 244-250.

Journal article
Published: 01 August 2014 in Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
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A novel technique, called Divergent, for single-tube real-time PCR genotyping of point mutations without the use of fluorescently labeled probes has recently been reported. This novel PCR technique utilizes a set of four primers and a particular denaturation temperature for simultaneously amplifying two different amplicons which extend in opposite directions from the point mutation. The two amplicons can readily be detected using the melt curve analysis downstream to a closed-tube real-time PCR. In the present study, some critical aspects of the original method were specifically addressed to further implement the technique for genotyping the DNM1 c.G767T mutation responsible for exercise-induced collapse in Labrador retriever dogs. The improved Divergent assay was easily set up using a standard two-step real-time PCR protocol. The melting temperature difference between the mutated and the wild-type amplicons was approximately 5°C which could be promptly detected by all the thermal cyclers. The upgraded assay yielded accurate results with 157pg of genomic DNA per reaction. This optimized technique represents a flexible and inexpensive alternative to the minor grove binder fluorescently labeled method and to high resolution melt analysis for high-throughput, robust and cheap genotyping of single nucleotide variations.

ACS Style

Fabio Gentilini; Maria E. Turba. Optimization of the Divergent method for genotyping single nucleotide variations using SYBR Green-based single-tube real-time PCR. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 2014, 766-767, 14 -18.

AMA Style

Fabio Gentilini, Maria E. Turba. Optimization of the Divergent method for genotyping single nucleotide variations using SYBR Green-based single-tube real-time PCR. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 2014; 766-767 ():14-18.

Chicago/Turabian Style

Fabio Gentilini; Maria E. Turba. 2014. "Optimization of the Divergent method for genotyping single nucleotide variations using SYBR Green-based single-tube real-time PCR." Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 766-767, no. : 14-18.

Journal article
Published: 01 August 2013 in The Veterinary Journal
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Two single tube real-time PCR methods were designed to genotype the mutation responsible for von Willebrand disease type I (von Willebrand factor c.7437G>A) in Doberman Pinscher dogs: (1) the Divergent PCR assay, which is a modification of the bi-directional PCR amplification of a specific allele (BI-PASA) technique, and (2) a minor groove binder (MGB) real-time PCR assay using fluorescently labelled probes. There was complete agreement between the genotypes determined using the two real-time PCR methods and the results of sequencing of PCR products generated by conventional PCR from genomic DNA purified from the blood of 27 Doberman Pinscher dogs. The Divergent PCR assay yielded reliable results with ≥ 6.4 ng genomic DNA per reaction and the MGB real-time PCR assay yielded reliable results with ≥ 150 pg genomic DNA per reaction. Both real-time PCR methods are suitable for routine genetic testing for the von Willebrand disease type I mutation using blood samples.

ACS Style

Fabio Gentilini; Maria E. Turba. Two novel real-time PCR methods for genotyping the von Willebrand disease type I mutation in Doberman Pinscher dogs. The Veterinary Journal 2013, 197, 457 -460.

AMA Style

Fabio Gentilini, Maria E. Turba. Two novel real-time PCR methods for genotyping the von Willebrand disease type I mutation in Doberman Pinscher dogs. The Veterinary Journal. 2013; 197 (2):457-460.

Chicago/Turabian Style

Fabio Gentilini; Maria E. Turba. 2013. "Two novel real-time PCR methods for genotyping the von Willebrand disease type I mutation in Doberman Pinscher dogs." The Veterinary Journal 197, no. 2: 457-460.

Comparative study
Published: 08 May 2013 in Veterinary and Comparative Oncology
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The conventional polymerase chain reaction (PCR)/sequencing methods may be poorly suited for the detection of somatic mutations in canine mast cell tumour (MCT) samples owing to limited sensitivity. This study was aimed at establishing novel and more sensitive methods, assessing their limit of detection and comparing their sensitivity with conventional methods.Two different ‘driver’ somatic mutations of c‐KIT, together with the wild‐type counterparts, were cloned in plasmids to prepare standard samples with known concentrations of mutated alleles in a background of wild‐type alleles; the plasmids standards were assayed using either conventional or novel, highly sensitive technique. Conventional PCR/sequencing showed a sensitivity of 50–20%. Conversely, all the novel methods obtained higher sensitivities allowed reaching as low as 2.5–1.2% of the mutated DNA.The study demonstrates that early conventional methods could likely have underestimated the prevalence of KIT mutations of MCTs, therefore affecting the assessment of their relevance in prognosis and tyrosine kinase inhibitor (TKI) treatment effectiveness.

ACS Style

F. Gentilini; V. Mantovani; M. E. Turba. The use of COLD-PCR, DHPLC and GeneScanning for the highly sensitive detection of c-KIT somatic mutations in canine mast cell tumours. Veterinary and Comparative Oncology 2013, 13, 218 -228.

AMA Style

F. Gentilini, V. Mantovani, M. E. Turba. The use of COLD-PCR, DHPLC and GeneScanning for the highly sensitive detection of c-KIT somatic mutations in canine mast cell tumours. Veterinary and Comparative Oncology. 2013; 13 (3):218-228.

Chicago/Turabian Style

F. Gentilini; V. Mantovani; M. E. Turba. 2013. "The use of COLD-PCR, DHPLC and GeneScanning for the highly sensitive detection of c-KIT somatic mutations in canine mast cell tumours." Veterinary and Comparative Oncology 13, no. 3: 218-228.

Journal article
Published: 30 March 2013 in Veterinary Immunology and Immunopathology
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Lymphoma is one of the most common forms of cancer in dogs as it is in humans but, unlike humans, the cure rates in canines are still very low. Despite the fact that high grade B-cell lymphomas are considered to be chemotherapy responsive, almost all treated dogs ultimately relapse and die due to the residual malignant lymphocytes, namely minimal residual disease (MRD). It would be extremely valuable for clinicians to detect, monitor and quantify MRD for risk group stratification, effective treatment intervention and outcome prediction. The PCRs targeting the Ig gene rearrangements constitute one of the most reliable tools to this end. We have recently validated a method which exploits hairpin-shaped primers for quantifying MRD. In the present study, that method is conveniently used for retrospectively monitoring MRD in the peripheral blood of 8 dogs diagnosed with B-cell lymphoma who underwent chemotherapy. All dogs attained complete remission. The median disease-free interval was 254.5 days (range 63–774) while the median survival time was 313.5 days (range 143–817 days). At admission, all dogs, except one which had already been treated with prednisone, had circulating neoplastic cells. All dogs attained complete remission (CR) which was almost always matched with a complete MRD response. The persistence of MRD despite apparent CR indicated a worse prognosis and a short duration of CR. Finally, the relapse is consistently anticipated by the reappearance of MRD in the peripheral blood. The study confirmed the suitability of an MRD monitoring assay as a clinical decision-making tool.

ACS Style

Fabio Gentilini; Maria E. Turba; Monica Forni. Retrospective monitoring of minimal residual disease using hairpin-shaped clone specific primers in B-cell lymphoma affected dogs. Veterinary Immunology and Immunopathology 2013, 153, 279 -288.

AMA Style

Fabio Gentilini, Maria E. Turba, Monica Forni. Retrospective monitoring of minimal residual disease using hairpin-shaped clone specific primers in B-cell lymphoma affected dogs. Veterinary Immunology and Immunopathology. 2013; 153 (3-4):279-288.

Chicago/Turabian Style

Fabio Gentilini; Maria E. Turba; Monica Forni. 2013. "Retrospective monitoring of minimal residual disease using hairpin-shaped clone specific primers in B-cell lymphoma affected dogs." Veterinary Immunology and Immunopathology 153, no. 3-4: 279-288.

Journal article
Published: 31 October 2012 in Research in Veterinary Science
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Procalcitonin (PCT), recognised as a marker of sepsis, was investigated in a porcine model of endotoxic shock. The results showed that continuous IV infusion (1–4 h) of LPS (40 μg/kg) in pigs was able to induce a generalised increase of PCT expression in lung, heart, kidney and liver. The increase in PCT was significant only in kidney and was accompanied by an increase in IL-6 gene expression. In vitro results demonstrated that peripheral blood mononuclear cells (PBMCs), as well as endothelial cells, were potentially capable of contributing to in vivo extrathyroidal PCT production. These findings support previous data from pigs concerning the occurrence of widespread activation of PCT extrathyroidal gene expression during endotoxic shock in pigs. Nevertheless, the levels of PCT detected were very low, suggesting the need for additional studies to validate the pig as a reliable animal model for investigating the role of PCT in sepsis.

ACS Style

Augusta Zannoni; Massimo Giunti; Chiara Bernardini; Fabio Gentilini; Andrea Zaniboni; Maria Laura Bacci; Monica Forni. Procalcitonin gene expression after LPS stimulation in the porcine animal model. Research in Veterinary Science 2012, 93, 921 -927.

AMA Style

Augusta Zannoni, Massimo Giunti, Chiara Bernardini, Fabio Gentilini, Andrea Zaniboni, Maria Laura Bacci, Monica Forni. Procalcitonin gene expression after LPS stimulation in the porcine animal model. Research in Veterinary Science. 2012; 93 (2):921-927.

Chicago/Turabian Style

Augusta Zannoni; Massimo Giunti; Chiara Bernardini; Fabio Gentilini; Andrea Zaniboni; Maria Laura Bacci; Monica Forni. 2012. "Procalcitonin gene expression after LPS stimulation in the porcine animal model." Research in Veterinary Science 93, no. 2: 921-927.

Case reports
Published: 05 September 2012 in Journal of Veterinary Diagnostic Investigation
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Neurolymphomatosis is a very rare form of nervous system infiltration by lymphoma that can affect cranial and peripheral nerves and spinal nerve roots. The clinical appearance can mimic autoimmune or paraneoplastic neuropathies. To date, only 2 cases of neurolymphomatosis have been reported in the veterinary literature (1 dog and 1 cat). A case of neurolymphomatosis in a 5-year-old female Domestic Shorthair cat is reported. Two, whitish, bosselated, non-symmetric masses (1 cm × 1.2 cm × 0.5 cm) that incorporated almost all cranial nerves and semilunar ganglia occupying the basisphenoid depression were histologically composed of a proliferation of monomorphic lymphocytes. These lymphoid cells were positive for CD3 (T-cell lymphoma). Nested polymerase chain reaction detected feline leukemia provirus. Fragment analysis of feline T-cell receptor (TCR) gene rearrangements evidenced an oligoclonal pattern with few peaks of similar height. The integration of pathologic with biomolecular findings adds to the information concerning the role of Feline leukemia virus on TCRγ rearrangements in cases of feline lymphoma.

ACS Style

Luciana Mandrioli; Maria Morini; Roberta Biserni; Fabio Gentilini; Maria Elena Turba. A case of feline neurolymphomatosis: pathological and molecular investigations. Journal of Veterinary Diagnostic Investigation 2012, 24, 1083 -1086.

AMA Style

Luciana Mandrioli, Maria Morini, Roberta Biserni, Fabio Gentilini, Maria Elena Turba. A case of feline neurolymphomatosis: pathological and molecular investigations. Journal of Veterinary Diagnostic Investigation. 2012; 24 (6):1083-1086.

Chicago/Turabian Style

Luciana Mandrioli; Maria Morini; Roberta Biserni; Fabio Gentilini; Maria Elena Turba. 2012. "A case of feline neurolymphomatosis: pathological and molecular investigations." Journal of Veterinary Diagnostic Investigation 24, no. 6: 1083-1086.

Evaluation study
Published: 01 August 2012 in Journal of Clinical Microbiology
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A fundamental role for the endosymbiotic bacteria Wolbachia pipientis in the pathogenesis of Dirofilaria immitis infections has emerged in recent years. Diagnostic opportunities arising from this breakthrough have not yet been fully exploited. This study was aimed at developing conventional and real-time PCR assays to carry out a molecular survey in a convenience sample of cats living in an area where D. immitis is endemic and to evaluate the detection of bacterial DNA in blood as a surrogate assay for diagnosing filaria-associated syndromes in cats. COI and FtsZ loci were used as targets for D. immitis and Wolbachia PCR assays, respectively, and real-time TaqMan PCR assays were used only for Wolbachia . A convenience sample of 307 disease-affected or healthy cats examined at a University facility were PCR tested, and their medical records were investigated. Conventional nested PCR for Wolbachia amplified the endosymbionts of both D. immitis and D. repens , while real-time PCR was highly specific only for the former. Observed prevalences of 0.3 and 10.4% were found using conventional nested PCR assays for D. immitis and real-time PCR for Wolbachia , respectively. Similar prevalences were established using the Wolbachia nested PCR (98% concordance with real-time PCR). The group of Wolbachia -positive samples had a significantly higher proportion of subjects with respiratory signs (29.0% versus 9.7%; P = 0.002). The findings of this study indicate that a highly sensitive PCR assay can be used to detect the Wolbachia organism in the peripheral blood of cats with respiratory signs.

ACS Style

Maria Elena Turba; Elisa Zambon; Augusta Zannoni; Samanta Russo; Fabio Gentilini. Detection of Wolbachia DNA in Blood for Diagnosing Filaria-Associated Syndromes in Cats. Journal of Clinical Microbiology 2012, 50, 2624 -2630.

AMA Style

Maria Elena Turba, Elisa Zambon, Augusta Zannoni, Samanta Russo, Fabio Gentilini. Detection of Wolbachia DNA in Blood for Diagnosing Filaria-Associated Syndromes in Cats. Journal of Clinical Microbiology. 2012; 50 (8):2624-2630.

Chicago/Turabian Style

Maria Elena Turba; Elisa Zambon; Augusta Zannoni; Samanta Russo; Fabio Gentilini. 2012. "Detection of Wolbachia DNA in Blood for Diagnosing Filaria-Associated Syndromes in Cats." Journal of Clinical Microbiology 50, no. 8: 2624-2630.